Tatiana G. Smirnova

Mycobacterium tuberculosis Type II Toxin-Antitoxin Systems: Genetic Polymorphisms and Functional Properties and the Possibility of Their Use for Genotyping

Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of Mycobacterium tuberculosis in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of M. tuberculosis was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of M. tuberculosis strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of M. tuberculosis. The possibility of using our set of genes for genotyping using PCR is also demonstrated.

Synthesis and activity of (+)-usnic acid and (−)-usnic acid derivatives containing 1,3-thiazole cycle against Mycobacterium tuberculosis

Abstract New usnic acid (UA) derivatives were investigated in vitro to elucidate their potential inhibitory activities on the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis. Seven pairs of enantiomers of thiazole UA derivatives were tested using the M. smegmatis strain mc2 155 test system, and the “structure–activity” relationship was established. The most active compounds were (+)-3 and (−)-3, and their kinase inhibitory activities were investigated. The results obtained using the Streptomyces lividans APHVIII+ and M. smegmatis APHVIII+ test systems indicated the significant protein kinase activity of these compounds and revealed the species specificity of the actions of the dextro- and levorotatory isomers. Both isomers, (+)-3 and (−)-3, possess similar inhibitory activity against M. tuberculosis H37Rv. The action of the isomers on eukaryotic cells was also investigated, and the results demonstrate that the dextrorotatory isomer (+)-3 leads to the lysis of intact macrophages to a degree higher than that obtained spontaneously and significantly higher than that obtained with the levorotatory isomer.

Resistance to pyrazinamide in Russian Mycobacterium tuberculosis isolates: pncA sequencing versus Bactec MGIT 960.

Resistance to pyrazinamide (PZA) may impact clinical outcome of anti-tuberculosis chemotherapy. PZA susceptibility testing using MGIT 960 is not reliable and little information is available on the prevalence of PZA resistance in Russia. A collection of 64 clinical isolates of Mycobacterium tuberculosis, including 35 multidrug resistant and extensively drug-resistant (MDR/XDR), was analyzed for PZA resistance using MGIT 960, Wayne test, and sequencing of PZA resistance genes pncA, rpsA and panD. In addition, we analyzed 519 MDR-TB strains for susceptibility to PZA by MGIT 960. Sequencing of pncA revealed 17 of 25 (68%) MDR strains and all 10 XDR strains harboring pncA mutations. A correlation of φ = 0.81 between MGIT 960 and pncA sequencing was observed. Mutations in rpsA and panD not associated with PZA resistance as defined by MGIT 960 were identified. We found 1 PZA-resistant strain without mutations in known PZA resistance genes. Almost 73% of MDR-TB strains isolated in Moscow, Russia, were PZA-resistant by MGIT 960 testing of 519 MDR-TB clinical isolates. Further studies are needed to determine the role of rpsA and panD mutations in possible low-level PZA resistance and to identify the molecular basis of new PZA resistance in the isolate without known PZA resistance mutations.

5‐Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis

Three series of 5‐arylaminouracil derivatives, including 5‐(phenylamino)uracils, 1‐(4′‐hydroxy‐2′‐cyclopenten‐1′‐yl)‐5‐(phenylamino)uracils, and 1,3‐di‐(4′‐hydroxy‐2′‐cyclopenten‐1′‐yl)‐5‐(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1‐(4′‐hydroxy‐2′‐cyclopenten‐1′‐yl)‐3‐(4‴‐hydroxy‐2‴‐cyclopenten‐1‴‐yl)‐5‐(4″‐butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug‐resistant strain MS‐115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

New 5-modified 2′-deoxyuridine derivatives: synthesis and antituberculosis activity

New 2′-deoxyuridine derivatives containing long-chain hydrophilic substituents or hydrophilic groups at the 5 position of uracil were synthesized. 5-[4-(2-Carboxyethylcarbonylamino)-butyloxymethyl]-2′-deoxyuridine completely inhibits the growth of Mycobacterium tuberculosis at a concentration of 50 μg mL−1. However, oligo(ethylene glycol) derivatives do not exhibit activity even at high concentrations.

New assay to diagnose and differentiate between Mycobacterium tuberculosis complex and nontuberculous mycobacteria

The purpose of the present study was to create a real-time PCR test system allowing simultaneous detection of nontuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) both in culture and sputum. NTM cultures (18 strains, 18 species), MTBC cultures (16 strains, 2 species) and non-mycobacterial microorganisms from the collection of the Central Research TB Institute (CTRI) were used for the preliminary evaluation of the test system. 301 NTM cultures from patients with mycobacteriosis were used to assess the sensitivity of the developed test system. Clinical respiratory samples (sputum) from 104 patients with mycobacteriosis, 3627 patients with tuberculosis and 118 patients with other lung diseases were used for diagnostic sensitivity and specificity testing. The specificity and sensitivity of the assay for MTBC was found to be 100% both in culture and sputum samples; for NTM, the specificity was 100% in culture and sputum, the sensitivity reached 100% in culture and 73.1% in sputum samples. Positive predictive value (PPV) and negative predictive value (NPV) of the assay for culture were both 100%, for clinical material 100% and 80.8%, respectively. The limit of detection at the probability of detection 95% (LoD95%) was estimated to be 16 cfu/ml for M. tuberculosis H37RV and 1200 cfu/ml for M. avium.

Dual-targeted anti-TB/anti-HIV heterodimers

&NA; HIV and M. tuberculosis are two intersecting epidemics making the search for new dual action drugs against both pathogens extremely important. Here, we report on the synthesis and suppressive activities of five dual‐targeted HIV/TB compounds. These compounds are heterodimers of AZT, as anti‐HIV molecules, and 5‐substituted uracil derivatives, as anti‐TB molecules. We found that these compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. We identified one particular heterodimer that inhibited both HIV and the drug‐resistant TB strain MS‐115 most potently. This compound demonstrated low toxicity and had no cytostatic effect on cells in culture, constituting an ideal candidate for future development and further in vivo testing.

[Synthesis and biological properties of α-thymidine 5'-aryl phosphonates].

Diethyl(N-arylaminocarbonyl)methyl phosphonates have been obtained by the reaction of diethylphosphonoacetic acid imidazolides with methyl-4-aminobenzoate or 3,5-bis(trifluoromethyl)phenylamine. Their treatment with Me3SiBr in DMF led to a mixture of the corresponding (N-arylaminocarbonylmethyl)phosphonic acids and their monoethyl esters. After separation, they were condensed with 3′-O-acetyl-α-thymidine, which, after the removal of the acetyl protecting group, gave (α-D-thymidine-5′-yl)-[4-aminocarbonyl-, methoxycarbonyl-, or carboxy)phenylaminocarbonyl]methyl phosphonates and (α-D-thymidine-5′-yl)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It was shown that the compounds are stable under different conditions, low toxic (in Vero and K-562 cell cultures), and capable of penetrating into K-562 cells. Only ethyl (α-D-thymidine-5′-yl)-[4-(methoxycarbonyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the growth of the laboratory strain M. tuberculosis H37Rv.